Although the atomic resolution structures of viruses represent well-resolved end-products, the process of virus assembly remains ill-defined with regard to its regulation within virus-infected cells and the specific biochemical properties of the viral structural proteins that determine the pathways of assembly. The purpose of this proposal is to use polyoma and papillomaviruses as model systems to study both the cell biology and biochemistry of virus assembly. We will study how and where these viruses are assembled. We hypothesize that cellular chaperone proteins, specifically the hsc70 family, maintain the viral capsid proteins in an unassembled form until they encounter viral genomes, which have been specifically marked by a nonstructural viral protein such as the polyoma large T-antigen or papilloma E2 protein bound to the viral DNA. By unknown mechanisms this encounter triggers the assembly of the capsid proteins around the viral minichromosome. We also hypothesize that specific locations within the cell nucleus, the PML-nuclear bodies, are the site of this interaction and subsequent virus assembly. In order to test these hypotheses we will use purified chaperone proteins and viral capsid proteins to study capsid assembly in vitro. We will attempt to encapsidate both naked DNA and minichromosome substrates with these proteins. Finally, we will characterize the PML-nuclear bodies during virus infection both by immunohistochemistry and cryo-electron microscopy. Structural, biochemical, and cell biological understanding of virus assembly provides a rational basis for developing anti-viral therapeutic agents, and have previously provided the scientific foundation for the new HPV vaccines that promise to prevent a majority of cervical cancers. In an era of immunosuppression secondary to transplantation regimens, cancer chemotherapy, and HIV infection, the human polyomaviruses such as JCV and BKV are now encountered as potential pathogenic agents, and new viruses such as the Merkel cancer-related polyomavirus are still being discovered. PUBLIC HEALTH RELEVANCE: Polyoma and papillomaviruses are infectious pathogens that have been linked to the development of human cancers. We are studying the way in which these viruses grow in cells in order to identify steps where therapies can be targeted to stop infection. Related previous studies have already led to the development of new papillomavirus vaccines that promise to prevent a majority of cervical cancers.